Mendelian randomization of gut microbiota and exploratory testing of preventive effect of commercial Rothia mucilaginosa in constipated mice

Abstract

Constipation is linked to gut microbiota dysbiosis, but causal microbial contributors and tractable bacterial interventions remain incompletely defined. This study first performed two-sample Mendelian randomization using Dutch Microbiome Project gut-microbiome Genome-Wide Association Study (GWAS) data ( N  = 7,738) and a European constipation GWAS ( N  = 411,623) to identify gut taxa linked to constipation. Then, in vitro metabolomics and a loperamide-induced constipation mouse model were used in male C57BL/6 mice to exploratorily test whether the commercial Rothia mucilaginosa strain BNCC363031 prevents loperamide-induced constipation. In an exploratory two-sample MR screen across 207 gut taxa, genetically predicted Rothia was nominally associated with lower constipation risk [odds ratio ( OR ) = 0.916, p  = 0.0041], and the species-level Rothia mucilaginosa showed a concordant nominal protective association ( OR  = 0.921, p  = 0.0232). In mice ( n  = 8 per group), preventive gavage partially restored loperamide-suppressed defecation: the cross-day mean fecal pellet count in the last 3 h per day was higher in the Rothia mucilaginosa group (RG) than in model controls (MCs) (7.94 vs. 4.41; p  = 0.0306) but lower than in normal controls (NCs) (13.16; p  = 0.00162), whereas body weight; small-intestinal transit; and immunohistochemical zonula occludens-1 (ZO-1), Claudin-1, and mucin 2 (MUC2) H-scores were unchanged. A fecal 16S V4 signal corresponding to R. mucilaginosa was detected at a very low abundance in only 2/8 RG mice; in vitro metabolomics identified candidates of secreted metabolites, including acetic acid. This study provides a reference for research on Rothia mucilaginosa in the context of constipation.